Abstract

Objective: The purpose of the present study was to evaluate the inflammatory response caused by three calcium silicate based cements in rat’s subcutaneous tissue. Materials and methods: Fifteen Wistar rats were divided into three groups of 1, 4, and 8 experimental weeks. Sterile silicone tubes were filled with Micro-Mega Mineral Trioxide Aggregate (MM-MTA), Biodentine (BD) and EndoSequence Root Repair Material (ESRRM) putty and implanted subcutaneously. Empty tubes were implanted as negative control. After the experimental periods, all animals were sacrificed and the specimens stained histologically with hematoxylin-eosin (H&E), Masson’s trichrome and Toluidine blue to assess the type of inflammation, intensity and extent of inflammatory cells, foreign body reaction, fibrous capsule thickness, coagulated necrosis, capillary reaction and vascular congestion, amount and the distribution of mature collagen and mast cell population. Statistical analysis was performed by the Kruskal-Wallis and Mann-Whitney U tests. The level of significance was 5% (P<0.05). Results: MM-MTA provoked severe inflammation after 1 week, which was significantly different from ESRRM putty and control (P<0.05); BD produced less biological reaction than MM-MTA and more than ESRRM and control during the initial periods. While both MM-MTA and BD had reduced inflammatory reaction with time. ESRRM showed tissue-tolerance features almost comparable to control during all experimental periods. Conclusion: ESRRM putty was significantly more biocompatible than MM MTA and BD in the 1st week of the experiment. However, there was no significant difference between the materials at the end of the 8th weeks. ESRRM putty, BD, and MM MTA can be considered suitable calcium silicate materials.

Keywords

Introduction

An ideal root repair material should be biocompatible, have the
ability to adhere to dentin, maintain a sufficient seal, be
insoluble in tissue fluids, be dimensionally stable, nonresorbable
over time, radiopaque, easily manipulated, adequate
compressibility, adequate working time, quick setting time, and
unaffected by blood contamination [1].

Biocompatibility of a root filling material, when used in pulp
capping, perforation repair, or as a retrograde filling, may
influence the viability of periradicular cells and cause cell
death by apoptosis or necrosis. To promote healing and
restoration of the function of the tooth, dental materials should
either stimulate cellular repair or be biologically neutral [2].

Mineral Trioxide Aggregate yields good biocompatibility
results; however, its handling and setting time are not ideal. MicroMega MTA (MM-MTA; MicroMega Besanchon,
France), another formulation of MTA is being developed to
overcome drawbacks of original MTA products in 2011. It is
injectable osteo-conductive, osteo-inductive and biocompatible
tricalcium silicate-based cement containing calcium carbonate,
which purportedly reduces the setting time [3-6].

Biodentine (BD) (Septodont, Saint Maur de Fosses; France)
has been developed as novel tricalcium silicate-based cement
in 2009 [7]. It was described as a bioactive dentin substitute
with apatite formation after immersion in phosphate solution
[8]. BD is reported as presenting better biological properties
than other tricalcium silicate cements such as MTA. Zhou et al.
[2] and Laurent et al. [9] assessed the cytotoxicity of
biodentine using human gingival fibroblast and pulp. Other
studies compared biocompatibility and gene expression of BD
and MTA using three-dimensional multicellular spheroid cultures and murine fibroblasts, they observed that BD was
non-cytotoxic and non-genotoxic [10,11].

An alternative material, EndoSequence Root Repair Materials
putty (ESRRM putty) was developed as a premixed, injectable
material formulated using bioceramic technology. It has been
released by Brasseler USA (Savannah, GA) to be used as a
clinical replacement for MTA. ESRRM putty has the
advantages of faster setting and superior handling properties
[12]. Alanezi et al. [13] Ma et al. [14] evaluated the
cytotoxicity of ESRRM putty using human ginigival fibroblast
and L929 mouse fibroblasts by MTT assay and they reported
similar in-vitro biocompatibility to MTA. On the other hand,
Martínez-Cortés et al. [15] found that ERRM material was
more biocompatible than MTA and did not exhibit cytotoxic
effects on mouse and human fibroblast.

The purpose of this study was to evaluate the biocompatibility
of three calcium silicate-based materials in rat’s subcutaneous
tissue using different special stains at different time periods.

Materials and Methods

This study performed in accordance with the principles of
laboratory animal care (NIH publication 85-23, 1985). The
national laws on animal use were also observed for the present
study by getting authorization from the Ethical Committee for
Animal Research of the Sulaimani University (No. 9,
6.2.2017).

Fifteen male Wistar albino rats aged (4-5) months and
weighted (250-350 g) were used in this study. The animals
were housed in temperature-controlled rooms (18-23ºC) and
were provided with water and food ad libitum. Animal care
was performed according to the Farabi Comprehensive Center
of Excellence in Ophthalmology, Tehran University of Medical
Sciences.

Forty-five silicone tubes of (7 mm length, inner diameter 2 mm
and the outer diameter of 4 mm with both open ends) were
filled with different tested materials and fifteen extra silicone
empty tubes were used as negative controls. Biodentine
(Septodont, Saint Maur de Fosses; France) was prepared
according to the manufacturer’s recommendations and inserted
into the tubes with amalgam carrier (Shanghai, China) and
condensed using small sized ash condenser (Shanghai, China).
EndoSequence Root Repair Material putty (BC Fast Set Putty)
(Brasseler USA; Savannah, GA) was available in a ready-touse
and was directly inserted into the silicone tubes. Micro
Mega MM-MTA was available in a capsule and it was
prepared according to the manufacturer ’ s instructions and
inserted into the tubes.

After administration of xylazine 2% (20 mg/kg Alfasan
Woerden-Holland, Holland) and ketamine 10% (100 mg/kg
Alfasan Woerden-Holland, Holland) intramuscular anesthesia,
the backs of the animals were shaved, antisepsis was obtained
with 5% iodine solution, and a 2.0 cm incision was formed in a head-tail orientation with #15 Bard-Parker blade (Moris,
Germany), creating two pockets on each side of the incision.
Three silicone tubes, containing the three tested materials, and
an empty tube, as the control, were implanted in each animal in
opposite directions [upper right (MM-MTA), upper left (empty
tube), lower right (ESRRM putty), and lower left (BD)]. The
skin was closed with a 4/0 silk suture (Lenosilk, Istanbul,
Turkey).

On 7 days, 4 weeks and 8 weeks after implantation, the
animals were euthanized by an anesthetic overdose and CO2.
Sixty samples including silicone tubes with the surrounding
tissues (20 samples for each time period) were removed and
fixed in 10% buffered formalin at pH 7.0 for 48 hours.

Histological preparation and staining

In the histological examination of the prepared samples, the
parameters were analyzed qualitatively and semi-quantitatively
[16]. The samples processed routinely, and three serial tissue
sections of 5 μm thickness were cut from each block. One
section stained with hematoxylin-eosin (H&E) and used for
histologic evaluation of the type and intensity of inflammation,
the presence of giant cells, fibroblastic (capsule), capillary
reactions, and coagulated necrosis. The remaining two sections
were stained with two special stains. Masson trichrome stain
was used for subjectively evaluating the amount and the
distribution of mature collagen surrounding the open ends of
the implanted tubes. While toluidine blue stain (Drury and
Wallington, 1980) used to observe the occurrence of mast cells,
their distribution and degranulation.

All slides were examined by Olympus light microscope at high
power fields (40X) linked with a digital camera (ToupTek,
ToupView, x86, 3.7.4183, 2014). The captured image were
transferred to a monitor and analyzed by image J software
(Image Processing and Analysis in Java; US National Institutes
of Health). Inflammatory and mast cells at the opening of the
tube were counted after dividing the field area by nine-squared
grid (Figure 1).

Figure 1. A photomicrograph of a subcutaneous tissue reaction at a tube opening showing the division of the filed area by a digital grid into nine
squares X400 ((A) H&E, (B) toluidine blue stain).

The type of inflammatory cells (polymorphonuclear cells and
mononuclear cells) were identified and counted by two
observers blindly for two times and the mean was calculated.

The intensity of inflammatory reactions were scored as
follows: 0: without inflammation, 1: <25 cell counts, 2: 25<cell
count<50, 3: 50<cell count 75, and 4: Over 75 cell counts [17].
The number of metachromatic mast cells were analyzed
quantitatively with purple violet granules was counted as
average per field. Data were organized on specific excel sheet
and statistically analyzed by SPSS software (version) using the
Kruskal-Wallis test and Mann-Whitney U tests at a
significance level of 5% (P<0.05). Fibrous capsule was
considered to be either thin or thick. The presence or absence
of suppuration or necrosis was recorded.

Results

The quantitative results of inflammatory and mast cells
counting at 1st, 4th and 8th weeks are presented in tables 1 and
2.

Table 2. Mean and Standard Deviation of the mast cell and significant differences among the studied groups within different experimental periods.

In the ESRRM putty group, the extent of cell infiltration was
significantly less compared to the other group near to the
control (P=0.029) on day 7. However, no statistical difference
(p>0.05) was observed among them in the 4th and 8th week
periods.

On the other hand, the number of the mast cells statistically no
significant (p>0.05) among the control, MM-MTA, BD and
ESRRM putty groups in the 1st and 4th week periods. In MMMTA,
the number of mast cells was significantly increased
(P=0.037) with other observations at 8th weeks.

Empty tube (Negative Control)

At day 7 there was edematous loose connective tissue, contain
few crossing collagen fibrils (faint blue by trichrome stain)
with fibroblast, few small round capillaries, and mild
inflammatory reaction, at the opening of the tube (Figure 2)
after 4th weeks and 8th weeks, a band of dense collagen fibers
(stained lighter with trichrome than collagen fibers in the
mature granulation tissue) and mature fibroblast. The adjacent granulation tissue contained elongated capillaries within a
fibrillar background (increased amount of collagen fibers that
stained dark blue and arranged in a mesh-like pattern) with no
evidence of inflammatory cells (Figure 2). Sometimes this
granulation tissue invaginated into the tube hole (Figure 2).
The tissues attached to silicone tube also showed a band of a
fibrous capsule with mature fibroblast (Figures 2).

At day 7, a severe packed chronic inflammatory cells
aggregation was seen within unorganized fibrous connective
tissue that contained many small capillaries with no evidence
of necrosis (Figure 3). Trichrome stain showed blue fibers
within the granulation tissue.

At 4th weeks, still the above granulation tissue contained
severe chronic inflammatory cells infiltration with few giant
cells. The tested material was extruded outside the tube.
Collagen fibers surrounded the reaction and the
collagen fibers by trichrome stain seemed to be more organized
in a parallel manner.

At 8th weeks, the fibrous capsule was more organization
containing fewer mature fibroblasts without increasing its
thickness. However, the thick capsule stained purple-violet
unlike the normal fibers in the surrounding connective tissue,
which take dark violet color. Occasionally giant cells were
seen. The tested material presented as inclusion bodies inside
the macrophages.

Biodentine

The histological sections for the samples containing biodentine
materials at 7 days showed thin layered of collagen fibrils and
several active fibroblasts (swollen large nucleus and abundant
cytoplasm) just ahead to the tube opening with mild
inflammatory cells and loose connective tissue containing
dilated large capillaries. At 4th weeks, the fibrous
capsules became thicker and containing many active
fibroblasts. At 8th weeks, the fibrous capsule
become even thicker (nearly twice that observed in 4th weeks),
and it still contained active fibroblasts. Few giant cells were
also seen.

EndoSequence Root Repair materials (ESRRM)
Putty

At 7th days, the interface tissue reaction showed a localized
coagulative necrosis (limited) containing fragments of cells,
nuclei and test material. There was no evidence of
an inflammatory response. Just above this necrotic layer, there
was a fibrogenic reaction with active fibroblast and small
capillaries (loose granulation tissue). The amount of necrosis
unrelated to the amount and size of tested material particles.
The tiny particles of tested materials were engulfed by
macrophages resulting in granulation tissue with minimum
inflammatory cells infiltrate.

After 4th weeks, the tissue reaction was minimum and no
evidence of necrosis. The connective tissue fibers were more
organized with minimum capillaries and many macrophages
engulfing the materials. After 8th weeks, the
material was surrounded by a dense bundle of the fibrous
capsule with active fibroblast and there was no
inflammatory cell infiltrate.

Discussion

Due to changes in chemical composition, setting and physical
properties of new bioceramic root repair materials, assessment
of body response to evaluate any biological and toxic effect
need to be tested carefully. Different methods are available to
assess the biocompatibility and the cytotoxicity of the dental
materials used in endodontics including cell lines tissue
reactions in animals and clinical trials in humans [2,14,18-20].
The in-vivo subcutaneous implantation method is the most
reliable and simple method that can be performed in animal
model studies. The responses of subcutaneous connective
tissues can be studied via histologic examinations based on
selected criteria that involve inflammatory and
histoimmunologic reactions, gene expression, and cytokine
production. Furthermore, such a method allows for evaluating
the thickness of the fibrous capsule, which is a useful marker
of inflammation and assisting the biocompatibility of various
materials [21].

To obtain a model resembles the root canal system and
simulate clinical condition, standardization of material-tissue
interface and stabilizes the material in place, a silicone tube
was selected for this study which is well tolerated by tissues
and easy to process histologically than polytetrafluoroethylene
or polystyrene ones [22,23].

No previous histological studies had examined a combination
of the three bioceramic root repair materials (MM‑MTA, BA,
and ERRM putty) to determine their degrees biocompatibility
or bioactivity in the animal model. Our study investigated the
severity of chronic inflammation, the density of mast cells
infiltration which plays a potent role in the early and late
immune and inflammatory responses [24,25] and collagenous
capsule.

A consequential relationship exists between the formation of
the fibrous capsule and material tolerability, since it reflects an
immune response which renders foreign bodies that have been
recognized as inoffensive to the biological system [26].

Thus, we compare the duration of irritability and bioactivity of
these tested materials when they come into contact with living
tissues after (1, 4 and 8 weeks) based on based on the
recommendations of the ISO to determine both short and longterm
inflammatory reaction [16].

In the present study, the deposition of collagen fibers that
become later a well-organized fibrous capsule in the control
group and all the three tested materials increased over time
agrees with Khalil et al. [27], Pinheiro et al. [28] Taha et al.
[29] findings. They stated that the deposition of a fibrous
capsule around the implanted material is an indication of tissue
tolerance. However, it is inconsistent with the observations of
other previous studies in which the authors proposed that the
biocompatibility of the material and its sign of inflammation
inversely related to the amount of fibrous capsule that develops
around the tested material [30,31].

The thickening in the fibrous capsule was noted in all
specimens although in different extent. It is attributable to enhanced collagen synthesis cytokines (Interleukin-1 [IL-1],
IL-4) and growth factors (Platelet-Derived Growth Factor
PDGF and Tumor Growth Factor TGF-ß) secreted by
leucocytes, similar to the negative control tube indicating the
tolerability of the tested materials by the tissue [32].

At the end of the experiment considering the long-term tissue
response and wound healing process to significant reduction in
the inflammation intensity moving towards the formation of
granulation tissue and fibrous encapsulation of the implanted
material observed in all tested materials suggesting a good
interaction between the material and the cells from the
surrounding tissue, which is a sign of good biocompatibility of
MM-MTA, BD and ESRRM putty.

In the study, the empty silicone tubes used in the control group
generated mild inflammatory reaction with few new capillaries
formation capillaries in the subcutaneous tissue and they
allowed for the formation of connective tissue at first week
with no inflammatory response and thicker fibrous capsule at
4th and 8th weeks, similar to the result previously reported
[33,34]. The presence of an early inflammatory response
associated to the surgical trauma after tube implantation that
leads to tissue disintegration and consequent infiltration of
inflammatory cells.

Studies that evaluate tissue responses to new MTA-based
materials are essential; since it has been shown that the
biocompatibility and bioactivity of MTA can be affected by the
addition of various components to the material [35,36]. The
findings of the present study showed that MM-MTA elicited
significantly more irritation than BD and ERRM putty at 7
days and 4 weeks, however, the severity of inflammation
decrease over the time with more organization of fibrous
capsule around the silicone tubes at the end of 8 weeks. These
results are consistent with findings of Simesk et al. [37] who
investigated the biocompatibility of MM-MTA in rat
subcutaneous tissue and concluded that BD was more
biocompatible than MM-MTA and Bioaggregate in the 1st
week of the experiment with no significant difference between
the materials at the end of the 45th day while Chang et al. [38]
reported in their study that both MM-MTA and Bioaggregate
exhibit similar biocompatibilities, inflammatory response, and
odontoblastic differentiation, compared with ProRoot MTA in
human dental pulp cells. Furthermore, Köseoğlu et al. [6]
observed in their in-vitro study in which they evaluated the
cytotoxicity and cell attachment of MM-MTA, BD, Endocem
and MTA Angelus that MM-MTA and BD had similar
biocompatibility and cytotoxicity.

High alkalinity, calcium ion release, heat release upon setting,
and stimulation of inflammatory cytokines (interleukin 1 and
interleukin 6) are those factors attributed to the initial powerful
tissue response to the implanted MM-MTA [39-41].

Furthermore, the chemical composition of MM-MTA, which
are based on Portland cement, a large amount of aluminum and
trace amounts of arsenic, beryllium, cadmium and chromium
were detected. Kum et al. [42] observed from their study in
tracing the metal contents of MM-MTA that arsenic concentration was about 1.76 ppm which may be responsible
for early severe inflammatory response in rat subcutaneous
implantation with MM-MTA.

Comparing BD group with the other groups in this study, a
moderate inflammatory process was observed at 7 days;
however, the inflammation was found in initial periods was
significantly reduced over the time. These results agree with
previous studies done by Mori et al. [43] Simesk et al. [37] to
assess the biocompatibility of BD after implantation in rat
subcutaneous tissue. They observed a moderate inflammatory
reaction that induced initially was reduced to a mild or nonsignificant
level with increasing time. Thus inflammatory
response in the early periods for BD may be due to the
presence of calcium chloride and a hydrosoluble polymer in
the liquids of BD to reduce setting time and water reducing
agent which was able to reduce the viability and cell binding
and produce an inflammatory reaction in dental pulps [44].

On the other hand, the differences in inflammatory reaction
between BD and MM-MTA explained by the presence of
radiopacifier. For MM-MTA, the radiopacifier is bismuth
oxide, whereas for BD, it is zirconium oxide which had no
cytotoxic effect on differentiated human cells and mouse
fibroblast [45-47].

Moreover, the biocompatibility and bioactivity of calcium
silicate-based cements are highly depended on the amount of
calcium release. Accordingly, materials with more calcium ion
release exhibit better biological properties [48]. Okiji et al. [49]
compared BD and white ProRoot MTA in terms of Ca ion and
Si ion uptake by adjacent root canal dentine and they observed
that both materials formed tag-like structures, but dentine
element uptake and Ca ion release was more prominent for BD
than MTA.

In the present study, MM-MTA and BD was considered
biocompatible because the injurious effect observed in the
initial period was significantly reduced over time and this is
supported by Silveira et al. [50] Love et al. [51] who stated that
the biocompatibility of the materials depends on the reduction
of inflammatory response in a reasonable amount of time to
non-significant levels.

Concerning the comparison of the ESRRMs, MM‑MTA and
BD with the control groups, ESRRMs showed a statistically
significant reduction in inflammatory infiltration by the 7th day
with no inflammatory response at the end of experiments.

Furthermore, the results of the present study agree with the
finding reported by Khalil et al. [27] where grey MTA
produced more extensive inflammatory reaction than ERRM
and more detached particles after 7 and 30 days. However, it
contradicts the results registered by Taha et al. [29]. They
concluded that the MTA produced significantly less irritation
than ESRRM at 1 and 3 weeks while confirmed more
biocompatibility of ESRRM than after 6 weeks.

The difference in results may be attributed to different
consistencies of putty and paste ESRRM. The putty used in
this study had less setting time from the paste which did not actually set at 4 hours. Ma et al. [14] reported slightly higher
cytotoxicity for the paste form of ESRRM compared to the
putty form.

The event of coagulative necrosis observed in ESRRM putty
group at 1 week revealed the ability of this material to induce
calcification and mineralization [52].

The differences in the inflammatory reaction were observed in
the present study in that the detached particles of MM-MTA
were surrounded by severing inflammatory infiltrate, whereas
the detached BD and ESRRM putty particles were surrounded
by less infiltrate and encapsulated by a dense fibrous capsule.
The differences in tissue response to MM-MTA, BD and
ESRRM putty may be attributed to the difference in
composition, consistency and setting reaction. MM-MTA
contain heavy metals and toxic elements that were released to
the surrounding tissue at the opening ends of the implanted
silicone tubes [42], which would explain the increase in the
mast cell number after 4 and 8 weeks in MM-MTA group that
led to the persistence of inflammation.

Unfortunately, there is no in-vivo study comparing the
biocompatibility and cytotoxicity between BD and ESRRM
putty. This research considers the first study evaluates the
tissue response to MM-MTA, BD and ESRRM putty together
in the subcutaneous tissue of the rat model.

Conclusion

Under the limitation of this study, one can conclude that:

• The ESRRM putty was more biocompatible and produced
less tissue irritation than MM-MTA and Biodentine during
all time of experiments.